Innate and adaptive immune responses are initiated by macrophage phagocytosis, a complex cellular pathway that is not yet fully understood. To investigate how phagosome maturation is governed, the bacterial pathogen Legionella pneumophila can be exploited as a genetic probe of macrophage function. Previous studies support the hypothesis that L. pneumophila blocks phagosome-lysosome fusion by expressing on its surface at least two activities that are developmentally regulated. Knowledge of when, how and where inhibitors of phagosome maturation are expressed by L. pneumophila will be applied to identify the corresponding molecules. Factors on the bacterial surface that block phagosome-lysosome fusion will be sought genetically by isolating and characterizing mutations that affect either persistence of dotA mutants in macrophages, lectin binding, or hydrocarbon binding, and biochemically by comparing the profiles of surface proteins and carbohydrates of virulent and avirulent strains. Factors expressed by post-exponential phase bacteria to block fusion with lysosomes will also be identified by loci whose constituitive expression rescues a regulatory mutant from lysosomal killing. By understanding how L. pneumophila evades clearance by macrophages, one can deduce how these phagocytes routinely engulf, digest, and display foreign material, then recruit specialists to eliminate potential threats. Because macrophages are central effector cells in both humoral and cell-mediated immunity, a molecular description of their membrane traffic is likely to provide a myriad of opportunities for improving delivery of therapeutics to prevent and treat a variety of human diseases.
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